C-reactive protein was first described by Tillett and Francis J. Exp. Med., 52, 561-71 (1930)! who observed that sera from acutely ill patients precipitated with the C-polysaccharide of the cell wall of Streptococcus pneumoniae. Others subsequently identified the reactive serum factor as protein, hence the designation "C-reactive protein."
C-reactive protein (CRP) is synthesized in the liver, and its concentration in serum may increase as much as 1,000-fold during the acute phase response. See Gewurz et al., Adv. Int. Med., 27, 345-372 (1982); Kushner, Ann. N.Y. Acad. Sci., 389, 39-48 (1982); Pepys et al., Adv. Immunol., 34, 141-212 (1983). Although the exact role of CRP in the acute phase response is not known, it is believed to play an important part in host defense. For instance, it has been reported that: (1) CRP binds phosphorylcholine, suggesting a role for CRP as an opsonin for microorganisms and damaged tissue that have exposed phosphorylcholine groups; (2) CRP binds chromatin, suggesting that CRP may act to scavenge chromatin released by cell lysis; (3) CRP neutralizes platelet activating factor, suggesting that CRP may function as a regulator of platelet and neutrophil activities; and (4) CRP complexed to certain other molecules or liposomes activates complement, suggesting that CRP may trigger the complement cascade. See Kaplan et al., J. Immunol., 112, 2135-2147 (1974); Volanakis et al., J. Immunol., 113, 9-17 (1974); Siegel et al., J. Exp. Med., 140, 631-47 (1974); Siegel et al., J. Exp. Med., 142, 709-21 (1975); Mold et al., J. Exp. Med., 154, 1703-1708 (1981); Narkates et al., Proc. N.Y. Acad. Sci., 389, 172-182 (1982); Nakayama et al., Clin. Exp. Immunol., 54, 319-326 (1983); Robey et al., J. Biol. Chem., 259, 7311-7316 (1984); Robey et al., J. Exp. Med., 161, 1344-56 (1985); Vigo, J. Biol. Chem., 260, 3418-3422 (1985); Shephard et al., Clin. Exp. Immunol., 63, 718-27 (1986); Horowitz et al., J. Immunol., 138, 2598-2603 (1987); Tatsumi et al., Clinica Chimica Acta, 172, 85-92 (1988); DuClos et al., J. Immunol., 141, 4266-4260 (1988); DuClos et al., J. Immunol., 146, 1220-1225 (1991); Xia et al., FASEB J., 6, 1344a (1992).
CRP is a pentamer which consists of five identical subunits, each having a molecular weight of about 23,500. The pentameric form of CRP is sometimes referred to as "native CRP."
In about 1983, another form of CRP was discovered which is referred to as "modified-CRP" or "mCRP". Modified-CRP has significantly different charge, size, solubility and antigenicity characteristics as compared to native CRP. Potempa et al., Mol. Immunol., 20, 1165-75 (1983). Modified-CRP also differs from native CRP in binding characteristics; for instance, mCRP does not bind phosphorylcholine. Id.; Chudwin et al., J. Allergy Clin. Immunol., 77, 216a (1986). Finally, mCRP differs from native CRP in its biological activity. See Potempa et al., Protides Biol. Fluids, 34, 287-290 (1986); Potempa et al., Inflammation, 12, 391-405 (1988).
The distinctive antigenicity of mCRP has been referred to as "neo-CRP." Neo-CRP antigenicity is expressed on:
1) denatured CRP prepared using suitable conditions (described below); PA0 2) the primary translation product of DNA coding for CRP (preCRP); and PA0 3) CRP immobilized on solid surfaces.
Potempa et al., Mol. Immunol., 20, 1165-75 (1983); Mantzouranis et al., Ped. Res., 18, 260a (1984); Samols et al., Biochem. J., 227, 759-65 (1985); Chudwin et al., J. Allergy Clin. Immunol., 77, 216a (1986); Potempa et al., Inflammation, 12, 391-405 (1988).
The neo-CRP antigenicity may be detected with antibodies. For instance, an antiserum made specific for neo-CRP can be used. See Potempa et al., Mol. Immunol., 24, 531-41 (1987). Alternatively, the unique antigenic determinants of mCRP can be detected with monoclonal antibodies. Suitable monoclonal antibodies are described in U.S. Pat. No. 5,272,257, published PCT application WO 91/00872 (published Jan. 24, 1991; corresponding to U.S. Pat. No. 5,272,257), Ying et al., J. Immunol., 143, 221-228 (1989), Ying et al., Immunol., 76, 324-330 (1992), and Ying et al., Molec. Immunol., 29, 677-687 (1992).
A molecule reactive with antiserum specific for neo-CRP has been identified on the surface of 10-25% of peripheral blood lymphocytes (predominantly NK and B cells), 80% of monocytes and 60% of neutrophils, and at sites of tissue injury. Potempa et al., FASEB J., 2, 731a (1988); Bray et al., Clin. Immunol. Newsletter, 8, 137-140 (1987); Rees et al., Fed. Proc., 45, 263a (1986). In addition, it has been reported that mCRP can influence the development of monocyte cytotoxicity, improve the accessory cell function of monocytes, potentiate aggregated-IgG-induced phagocytic cell oxidative metabolism, and increase the production of interleukin-1, prostaglandin E and lipoxygenase products by monocytes. Potempa et al., Protides Biol. Fluids, 34, 287-290 (1987); Chu et al., Proc. Amer. Acad. Cancer Res., 28, 344a (1987); Potempa et al., Proc. Amer. Acad. Cancer Res., 28, 344a (1987); Zeller et al., Fed. Proc., 46, 1033a (1987); Potempa et al., Inflammation, 12, 391-405 (1988); Chu et al., Proc. Amer. Acad. Cancer Res., 29, 371a (1988). Chudwin et al., J. Allergy Clin. Immunol., 77, 216a (1986) teaches that mCRP can have a protective effect in mice challenged with gram-positive type 7F Streptococcus pneumoniae.
Other activities of mCRP have been discovered and are described in certain issued U.S. patents, copending U.S. applications and published PCT applications. In particular, it has been discovered that mCRP binds immune complexes and aggregated immunoglobulin and can, therefore, be used to remove immune complexes and aggregated immunoglobulin from fluids and to quantitate immune complexes. See published PCT application WO 89/09628 (published Oct. 19, 1989), which corresponds to co-pending U.S. application Ser. No. 08/271,137, filed Jul. 6, 1994 (which was a continuation of application Ser. No. 07/582,884, filed Oct. 3, 1990, now abandoned, which was a continuation-in-part of application Ser. No. 07/176,923, filed Apr. 4, 1988, now abandoned). Modified-CRP has also been found to be effective in treating viral infections (see co-pending U.S. application Ser. No. 08/117,874, filed Sep. 7, 1993, a continuation of application Ser. No. 07/799,448, filed Nov. 27, 1991, now abandoned), non-Streptococcal bacterial infections and endotoxic shock (see allowed U.S. application Ser. No. 07/800,508, filed Nov. 27, 1991), and cancer (see issued U.S. Pat. No. 5,283,238 and co-pending U.S. application Ser. No. 08/149,663, filed Nov. 9, 1993).
For a brief review of CRP and mCRP, see Gotschlich, Ann. N.Y. Acad. Sci., 557, 9-18 (1989). Kilpatrick and Volanakis, Immunol. Res., 10, 43-53 (1991) provides a recent review of CRP.
Prior to the present invention, mCRP was preferably made using purified CRP as a starting material. Generally, mCRP was prepared from CRP by denaturing the CRP. For instance, CRP could be denatured by: (1) treatment with an effective amount of urea (preferably 8M) in the presence of a conventional chelator (preferably ethylenediamine tetraacetic acid (EDTA) or citric acid); (2) adjusting the pH of the CRP to below about 3 or above about 11-12; or (3) heating CRP above 50.degree. C. for a time sufficient to cause denaturation (preferably at 63.degree. C. for 2 minutes) in the absence of calcium or in the presence of a chelator such as those listed above. Urea treatment has been the preferred method. In addition, mCRP can be prepared from CRP by adsorbing the CRP onto solid surfaces. It is believed that mCRP prepared from CRP is formed by the dissociation of the five CRP subunits, each of which then undergoes a spontaneous conformational change to form mCRP. See Bray et al., Clin. Immunol. Newsletter, 8, 137-140 (1987).
Although biological sources of CRP and methods of purifying it from those sources are well known, purified CRP can be obtained from such sources only in limited quantities. Accordingly, mCRP could not be produced from CRP in commercial quantities.
Genomic and cDNA clones coding for human, mouse, and rabbit CRP have been isolated. Tucci et al., J. Immunol., 131, 2416-2419 (1983); Whitehead et al., Science, 221, 69-71 (1983); Lei et al., J. Biol. Chem., 260, 13377-83 (1985); Woo et al., J. Biol. Chem., 260, 13384-88 (1985); Hu et al., Biochem., 25, 7834-39 (1986); Samols and Hu, Protides Biol. Fluids, 34, 263-66 (1986); Syin et al., J. Biol. Chem., 261, 5473-79 (1986); Ciliberto et al., Nucleic Acids Res., 15, 5895 (1987); Hu et al., J. Biol. Chem., 263, 1500-1504 (1988); Whitehead et al., Biochem. J., 266, 283-90 (1990). To obtain pentameric native CRP, eukaryotic host cells, preferably mammalian host cells, should be used. See Samols and Hu, Protides Biol. Fluids, 34, 263-66 (1986); Hu et al., J. Biol. Chem., 263, 1500-1504 (1988). Thus, native CRP could be produced in large quantities by recombinant DNA techniques and then converted into mCRP as described above. However, it would be convenient to have a direct method of making mCRP or a molecule having the biological activities of mCRP by recombinant DNA techniques.
As noted above, the primary translation product of the CRP mRNA (preCRP) has been found to express neo-CRP antigenicity. preCRP is a precursor protein consisting of a signal or leader sequence attached to the N-terminus of the CRP subunit. During normal processing, the signal or leader sequence is cleaved from the preCRP molecule to produce mature CRP subunits which assemble into pentameric native CRP. Accordingly, mCRP could be prepared by selecting conditions so that pentameric native CRP is not formed from the preCRP. This can be accomplished by expressing a CRP genomic or cDNA clone in a prokaryotic host. See Samols and Hu, Prot. Biol. Fluids, 34, 263-66 (1986).
In attempting to produce mCRP in this manner, Applicants have discovered that the product of a CRP cDNA clone expressed in Escherichia coli consists of aggregates of CRP subunits and/or preCRP and CRP fragments, as well as free CRP subunits and/or preCRP. This cDNA product is extremely insoluble, and purification has proved problematical. In particular, Applicants have discovered that a substantial proportion of the aggregates in these preparations are formed by covalent cross-linking, and such cross-linked aggregates must be discarded, thereby significantly reducing yields.
Therefore, being able to produce a mutant CRP subunit or preCRP molecule having the biological activities of mCRP, but which was less likely to form covalently cross-linked aggregates than the unmutated protein, would be highly desirable in order to make processing and purification easier and more efficient. The present invention provides mutant proteins having these characteristics, and these mutant proteins may be produced by site-directed mutagenesis of a CRP cDNA or genomic clone as further described below.
Agrawal et al., FASEB J., 6, 1427a (1992) and Agrawal et al., J. Biol. Chem., 267, 25352-58 (1992) report the use of site-specific mutagenesis of a CRP cDNA clone to investigate the structural determinants of the phosphorylcholine binding site of CRP. Eight mutant recombinant CRP's were prepared: Tyr40.fwdarw.Phe; Glu42.fwdarw.Gln; Tyr50.fwdarw.Phe and Glu42.fwdarw.Gln; Lys57.fwdarw.Gln; Arg58.fwdarw.Gly; Lys57.fwdarw.Gln and Arg58.fwdarw.Gly; Trp67.fwdarw.Lys; and Lys57.fwdarw.Gln, Arg58.fwdarw.Gly and Trp67.fwdarw.Lys. The authors concluded that Trp67 is critical for the structure of the phosphorylcholine binding site of CRP, that Lys57 and Arg58 also participate in the formation of this binding site, and that the tetrapeptide 39-Phe-Tyr-Thr-Glu has only a minimal or no role in the formation of this binding site.
Agrawal et al., J. Immunol., 152, 5404-5410 (1994) reports the use of site-specific mutagenesis of a CRP cDNA clone to investigate the structural determinants of the C1q binding site of CRP. Eleven mutant recombinant CRP's were prepared: Asp112.fwdarw.Asn; Asp112.fwdarw.Ala; Asp112.fwdarw.Lys; Asp112.fwdarw.Glu; Lys114.fwdarw.Thr; Lys114.fwdarw.Ala; Lys114.fwdarw.Glu; Lys114.fwdarw.Arg; Arg116.fwdarw.Leu; Asp112.fwdarw.Asn and Lys114.fwdarw.Thr; and Asp112.fwdarw.Asn and Arg116.fwdarw.Leu. The authors concluded that Asp112 plays a major role in the formation of the C1q binding site of CRP and that Lys114 and, to a lesser extent, Arg116 play important, but indirect, roles in C1q binding and activation of complement by CRP complexes.